The goal of this project is to discover novel broad-spectrum antibiotics acting against critically important human pathogens. The unmet need is especially acute for Gram negative pathogens, where we are rapidly running out of options for treating multidrug resistant species of Pseudomonas, Acinetobacter, Burkholderia, Klebsiella and Enterobacter. This is a challenging goal, since the last novel class of broad-spectrum antibiotics was discovered 40 years ago. NovoBiotic has a proprietary technology capable of meeting this challenge. We will obtain new antimicrobials from a previously inaccessible source - uncultured microorganisms that make up 99% of the total microbial diversity. In Phase I, we developed a trap for specific capture and growth of uncultured Actinomycetes. Most antibiotics derive from culturable representatives of this group of bacteria. The trap is a sterile slice of agar sandwiched between semi-permeable membranesthat is inserted into soil. Actinomycetes produce hyphae which enable them to penetrate into the trap through the pores in the membrane and form pure colonies. Many uncultured species that initially grow in situ are then capable of growing under regular conditions in vitro. The aims of Phase I were to assemble a collection of 1,000 novel Actinomycetes using the trap method, screen them for antimicrobial activity and dereplicate 50 of them. We achieved these milestones ahead of schedule and have in place a robust technology that will enable us to discover broad-spectrum antibiotics in this Phase II project. Half of the Actinomycetes captured by the trap are new, an impressive indication of the usefulness of this method. Of the 92 compounds that were dereplicated, we discovered two new antibiotics, one of which belongs to a novel class, glycosylated macrolactams. This suggests a probability of discovery ~10-2, compared to the current estimate of 10-7 for novel compounds from conventional culturable strains. The hit rate for compounds with broad-spectrum activity from this pilot library was 1%, and 1 of these 12 dereplicated antimicrobials is a potentially novel antibiotic. These findings strongly suggest that an expanded discovery effort in Phase II will lead to a considerable number of new broad-spectrum antimicrobials. In this project, we will screen up to 750,000 trap isolates, which should produce ~30 new broadspectrum compounds. There in vitro validation and structure determination will be followed by evaluation for toxicity in an animal model, and for efficacy in a murine model of K. pneumonia pulmonary infection. The end result of Phase II will be 2-3 validated novel broad-spectrum leads. These will be developed into drugs in the next phase of the program. PUBLIC HEALTH RELEVANCE: Bacteria that cause infections in the community and in hospitals are becoming increasingly resistant to currently available antibiotics;therefore there is an urgent need for new antibiotics that will ensure public health in the years to come. The purpose of this project is to employ a new technology to discover the next generation of effective antibiotics. These antibiotics will also provide the public with a stronger defense against the threat of bioterror.